Recombinant raccoon pox virus comprising the DNA encoding the nucleocapsid protein of feline infectious peritonitis virus

Wasmoen , et al. August 12, 1

Patent Grant 5656275

U.S. patent number 5,656,275 [Application Number 08/480,882] was granted by the patent office on 1997-08-12 for recombinant raccoon pox virus comprising the dna encoding the nucleocapsid protein of feline infectious peritonitis virus. This patent grant is currently assigned to American Home Products Corporation. Invention is credited to Lloyd Chavez, Hsien-Jue Chu, Terri Wasmoen.


United States Patent 5,656,275
Wasmoen ,   et al. August 12, 1997

Recombinant raccoon pox virus comprising the DNA encoding the nucleocapsid protein of feline infectious peritonitis virus

Abstract

This invention provides a recombinant raccoon poxvirus that expresses the nucleocapsid and transmembrane proteins of Feline Infectious Peritionitis Virus. The recombinant viruses are useful as vaccines, either alone or in combination with carriers and adjuvants.


Inventors: Wasmoen; Terri (Fort Dodge, IA), Chavez; Lloyd (Fort Dodge, IA), Chu; Hsien-Jue (Fort Dodge, IA)
Assignee: American Home Products Corporation (Madison, NJ)
Family ID: 22420074
Appl. No.: 08/480,882
Filed: June 7, 1995

Related U.S. Patent Documents

Application Number Filing Date Patent Number Issue Date
125516 Sep 22, 1993

Current U.S. Class: 424/199.1; 424/221.1; 424/211.1; 424/208.1; 424/274.1; 435/235.1; 435/320.1; 424/263.1; 424/216.1; 424/233.1; 424/202.1; 424/201.1; 424/819; 424/232.1; 424/207.1; 424/229.1
Current CPC Class: A61P 31/12 (20180101); C07K 14/005 (20130101); C12N 15/86 (20130101); A61K 39/00 (20130101); C12N 2770/20022 (20130101); C12N 2710/24022 (20130101); Y10S 424/819 (20130101); C12N 2710/24143 (20130101); C07K 2319/00 (20130101)
Current International Class: C12N 15/863 (20060101); C07K 14/165 (20060101); C07K 14/005 (20060101); C07K 14/065 (20060101); A61K 39/00 (20060101); A61K 039/295 (); A61K 039/275 (); A61K 039/215 (); A61K 039/21 (); A61K 039/265 (); A61K 039/118 (); C12N 015/00 (); C12N 007/00 (); A61K 039/23 (); A61K 039/155 (); A61K 039/125 ()
Field of Search: ;424/199.1,232.1,221.1,202.1,201.1,819,207.1,233.1,211.1,119.1,232,216.1,208.1 ;435/320.1,235.1

References Cited [Referenced By]

U.S. Patent Documents
5242686 September 1993 Chu et al.
5266313 November 1993 Esposito et al.
Foreign Patent Documents
0376744 Mar 1990 EP
Primary Examiner: Caputa; Anthony C.
Attorney, Agent or Firm: Darby & Darby

Parent Case Text



This is a continuation of application Ser. No. 08/125,516, filed Sep. 22, 1993, now abandoned.
Claims



What is claimed is:

1. A recombinant raccoon poxvirus having at least one internal gene comprising a DNA sequence encoding the nucleocapsid (N) protein of Feline Infectious Peritonitis Virus (FIPV).

2. The recombinant raccoon poxvirus of claim 1 wherein said internal gene has the amino acid sequence of SEQ ID NO: 6.

3. A vaccine comprising:

a recombinant raccoon poxvirus having at least one internal gene comprising a DNA sequence encoding the nucleocapsid (N) protein of Feline Infectious Peritonitis Virus (FIPV), and

a pharmaceutically acceptable carrier.

4. The vaccine of claim 3 wherein said internal gene has the amino acid sequence of SEQ ID NO: 6.

5. The vaccine of claim 3 further comprising inactivated or attenuated viruses selected from the group consisting of feline leukemia virus, feline panleucopenia virus, feline rhinotracheitis virus, feline calicivirus, feline immunodeficiency virus, feline herpesvirus, feline enteric coronavirus, and mixtures thereof.

6. The vaccine of claim 3 further comprising organisms selected from the group consisting of inactivated or attenuated feline Chlamydia psittaci, Microsporum canis, and mixtures thereof.

7. The vaccine of claim 3, further comprising a pharmaceutically acceptable adjuvant.

8. A method for preventing disease caused by Feline Infectious Peritonitis Virus (FIPV), comprising administering to a feline in need of such treatment via a subcutaneous or intramuscular route a vaccine comprising a recombinant raccoon poxvirus having at least one internal gene comprising a DNA sequence encoding the nucleocapsid (N) protein of FIPV.

9. The method of claim 8 wherein said internal gene has the amino acid sequence of SEQ ID NO: 6.
Description



FIELD OF THE INVENTION

The present invention pertains to the prophylaxis of disease caused by feline infectious peritonitis virus (FIPV), using recombinant raccoon pox viruses (RRPVs) expressing the nucleocapsid and transmembrane proteins of FIPV as vaccines.

BACKGROUND OF THE INVENTION

Feline infectious peritonitis virus (FIPV) induces a systemic infection in cats that is often fatal. The effusive form of the disease is characterized by accumulation of fibrinous ascitic fluid. The non-effusive form of the disease is characterized by granulomatous lesions in multiple organs including, but not limited to, liver, spleen, kidneys, lung, and intestines. Reviewed in Barlough, J. E. and C. A. Stoddart. Feline Coronaviral Infections in C. E. Greene (Ed.). Infectious Diseases of the Dog and Cats. W. B. Saunders Co., Philadelphia, Pa., 1990, pp. 300-312.

Feline infectious peritonitis virus is a coronavirus composed of three major structural proteins: The S (spike) protein, the E1 or M (transmembrane) protein, and the N (nucleocapsid) protein. Venema et al., Virology 181: 327-335, 1991 and Dale, et al., EPO 0,376,744.

Prior vaccines intended to prevent FIPV infection have actually been shown to exacerbate the disease caused by this virus. Pedersen, N. C. and J. W. Black, Am. J. Vet. Res. 44: 229-234, 1983; Vennema H., et al., J. Virol. 64: 1407-1409, 1990; Barlough, J. E., Can. J. Comp. Med. 49: 303-307, 1985; Barlough J. E. et al., Lab. Anim. Sci. 34: 592-597, 1984; Stoddart, C. A., et al., Res. Vet. Sci. 45: 383-388, 1988; and Pedersen, N. C., Adv. Vet. Sci. Comp. Med. 33: 413-428, 1989. This phenomenon apparently reflects an immune enhancement of infection mediated by immunoglobulins produced in response to the virus, in particular by those antibodies directed against the S protein. Olsen C. W. et al., J. Virol. 4: 175-189, 1981. Therefore, the best candidate vaccine for prophylaxis of this disease would be a preparation that induces strong cell-mediated immunity in the absence of enhancing antibodies. This could be accomplished with a vaccine that lacks the outer envelope protein but contains the other structural proteins of FIPV (N and E1). Prior attempts to vaccinate cats with a recombinant vaccinia virus expressing the N or E1 proteins of FIPV, however, have failed to induce strong protective immunity. Venema et al., Virology 181:327-335, 1991 and Dale, et al., European Patent Application 0,376,744. See also, Venema, European Patent Application 0,411,684.

What is needed in the art, therefore, is an effective vaccine against FIPV that utilizes the N and E1 proteins, or segments therefrom, as immunogens.

SUMMARY OF THE INVENTION

The present invention pertains to the induction of protective immunity to FIPV in cats. One object of the invention is to provide recombinant raccoon poxviruses containing the genes for the FIPV N or M/E1 proteins (RRPV-N and RRPV-E1, respectively).

A further object of the invention is to provide a feline vaccine comprising RRPV-N or RRPV-E1, either singly or in combination, or in combination with other viruses, bacteria, or fungi that have been inactivated or attenuated.

A still further object of the invention is to provide a method for preventing disease caused by FIPV, by administering to a feline in need of such treatment a vaccine comprising RRPV-N, RRPV-E1, or combinations thereof.

These and other objects and advantages, which will be apparent from this specification, are achieved by the invention described below.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A through 1E, when joined at respective match lines A--A through D--D, illustrate the nucleotide and amino acid sequence of the FIPV E1 protein and the (SEQ ID NO: 1) and (SEQ ID NO: 5), respectively. (FIGS. 1A and 1B, respectively.)

FIGS. 2A through 2G, when joined at respective match lines A--A through F--F, illustrate the nucleotide and amino acid sequence of the FIPV N protein (SEQ ID NO: 2) and (SEQ ID NO:6), respectively.

FIGS. 3A and 3B illustrate the plasmid used to clone the genes encoding the FIPV E1 and N proteins.

FIGS. 4A schematically shows the pSC11 transfer plasmid used to create RRPVs encoding the FIPV E1 protein.

FIGS. 4B schematically shows the pSC11 transfer plasmid used to create RRPVs encoding the N protein.

FIGS. 5A to 5F, when joined at respective match lines A--A through E--E, illustrate the nucleotide sequence of pSC11 FIPV E1.

FIGS. 6A through 6F, when joined at respective match lines A--A through E--E, illustrate the nucleotide sequence of pSC11 FIPV N.

FIG. 7 is a photograph of an ethidium bromide-stained agarose gel showing the analysis of RRPV-FIPV N and RRPV-FIPV E1 by polymerase chain reaction.

FIG. 8 is an imunoblot illustrating the detection of FIPV N and E1 proteins in virally infected cell lysates.

DETAILED DESCRIPTION OF THE INVENTION

The vaccine of the present invention may be prepared by creating recombinant raccoon poxviruses (RRPVs) containing the genes encoding the N or E1 proteins of FIPV or immunogenic fragments thereof. These genes are first inserted into a transfer plasmid, which is then introduced into appropriate host cells that have been previously infected with a raccoon poxvirus. As a result, the DNA from the transfer plasmid is incorporated into the poxvirus DNA by homologous recombination, producing the RRPVs that are released from the cells.

DNA encoding the FIPV N or E1 proteins is inserted into the transfer plasmid immediately downstream of a poxvirus promoter. In a preferred embodiment, the early/late 7.5 Kd protein promoter of vaccinia virus is used; however, alternate promoter elements that are functional in poxviruses can also be used.

The preferred transfer plasmid also contains a beta-galactosidase marker gene, which allows for selection and detection of the plasmid DNA sequences in recombinant viruses. It will be obvious to one skilled in the art that alternative selectable marker genes, such as the neomycin resistance gene or the E. coli gpt gene or others, can be used to practice the invention. Flanking the foreign gene of interest and the selectable marker gene are thymidine kinase DNA sequences, which facilitate recombinatorial integration of the plasmid DNA sequences into the raccoon poxvirus DNA.

Recombinant viruses expressing the FIPV N or E1 genes are prepared by first infecting a susceptible cell line such as Vero (ATCC CCL 81), BSC-1 (ATCC CCL 26), RAT-2 (ATCC CRL 1764), or CRFK (ATCC CCL 941) with wild type raccoon poxvirus (ATCC VR-838 or similar isolates, such as, for example, RCNV71-I-85A). Transfer plasmid DNA containing the E1 or N gene is then transfected into the infected cells using cationic liposome-mediated transfection, or other suitable techniques such as electroporation or calcium phosphate precipitation. Virus replication is allowed to proceed until cytopathic effects are noted in all cells.

Incorporation of the FIPV E1 or N genes into poxvirus DNA is accompanied by disruption of the viral thymidine kinase gene. Therefore, virus harvested from this infection may be isolated by selecting for the absence of a thymidine kinase gene; this is achieved by growth on tk-RAT-2 cells (ATCC CRL 1764) in the presence of 5-bromodeoxyuridine. Viruses containing a gene insert from the transfer plasmid are further identified by the appearance of a blue plaque color when grown in the presence of a chromogenic substrate for beta-galactosidase such as X-gal.

Viral plaques that survive these selection and screening procedures are then subjected to several cycles of plaque purification. Subsequently, the presence of the E1 or N genes is confirmed by polymerase chain reaction technology, and the presence of E1 or N protein is confirmed by immunoblot analysis using specific antibodies. These viruses are designated RRPV-FIPV E1 and RRPV-FIPV N, respectively.

In a further embodiment of the present invention, the genes encoding N and E1 were inserted into a single transfer plasmid. Introduction of this plasmid into cells previously infected with wild-type raccoon poxvirus results in the production of recombinant viruses that express both proteins simultaneously (RRPV-FIPV E1/N).

In a still further embodiment, RRPVs can be produced that express less-than-full-length segments of the FIPV E and N proteins. The techniques used to engineer transfer plasmids encoding partial sequences of E1 and N are well-known and widely used in the art, as are the methods for production and screening of RRPVs as detailed in this specification. For example, introduction of oligonucleotides containing a stop codon at various points along E1 or N DNA will produce a nested set of carboyxterminal-truncated versions of that gene, which can then be incorporated into RRPVs. It will be apparent to one of ordinary skill in the art that systematic screening of such recombinant RRPVs can establish whether the intact protein, or subfragments thereof, are most preferred in practicing the present invention. Furthermore, as stated above, DNA encoding different fragments of E1 and N can be used in a combination vaccine after incorporation into the same, or different, RRPVs.

For vaccine preparation, susceptible cells such as those listed above are infected with RRPVs at a multiplicity Of infection (MOI) of 0.1 infectious units/cell or less. In this specification, an infectious unit is defined as a Tissue Culture Infectious Dose (TCID.sub.50), an amount of virus yielding 50% infection under defined conditions. A method for TCID.sub.50 determination is detailed in Example 1 below. When cytopathology is noted in >90% of the cells, the infected cells and extracellular fluids (both of which contain viruses) are harvested as a single virus-cell lysate.

The highly concentrated virus stock to be used as a vaccine may be stored frozen (-50.degree. C. or colder) or lyophilized until the time of use. Compounds such as NZ-amine, dextrose, gelatin or others designed to stabilize the virus during freezing and lyophilization may be added. The virus initially present in the virus-cell lysate may be further concentrated using commercially available equipment.

Typically, the concentration of virus in the vaccine formulation will be a minimum of 10.sup.6.5 TCID.sub.50 per dose, but will typically be in the range of 10.sup.7.0 to 10.sup.9.0 TCID.sub.50 per dose. At the time of vaccination, the virus is thawed (if frozen) or, if lyophilized, is reconstituted with a physiologically-acceptable carrier such as deionized water, saline, phosphate buffered saline, or the like.

The present invention is not limited to native (i.e. replication-competent) RRPVs. The virus-cell lysate can be subjected to treatments commonly used in the art to inactivate viruses. A composition comprising inactivated virus and expressed protein will be effective in eliciting protective immunity against FIPV if it contains a sufficient quantity of FIPV protein. This type of vaccine would provide added assurance that recipient felines will not be exposed to infectious FIPV as a consequence of vaccination. In addition, a physiologically-acceptable adjuvant may be added to the virus, such as EMA 31 (Ethylene Maleic anhydride 31) (Monsanto Co., St. Louis, Mo.), NEOCRYL (Polyvinyl Chemical Industries, Wilmington, Mass.), MVP (Modern Veterinary Products, Omaha, Nebr.), Squalene, PLURONIC L121 (polyoxypropylene-polyoxyethylene block copolymer) or the like.

Individual raccoon poxviruses expressing the N or E1 genes may be mixed together for vaccination. Furthermore, the virus may be mixed with additional inactivated or attenuated viruses, bacteria, or fungi such as feline leukemia virus, feline panleukopenia virus, feline rhinotracheitis virus, feline calicivirus, feline immunodeficiency virus, feline herpesvirus, feline enteric coronavirus, feline Chlamydia psittaci, Microsporum canis, or others. In addition, antigens from the above-cited organisms may be incorporated into combination vaccines. These antigens may be purified from natural sources or from recombinant expression systems, or may comprise individual subunits of the antigen or synthetic peptides derived therefrom.

In a further embodiment of the present invention, live or inactivated RRPV virus-cell lysates can be incorporated into liposomes, or encapsulated in peptide-, protein-, or polysaccharide-based microcapsules prior to administration, using means that are known in the art.

The final vaccine is administered to cats in a volume that may range from about 0.5 to about 5 ml. The vaccine can be administered by subcutaneous, intramuscular, oral intradermal, or intranasal routes. The number of injections and their temporal spacing may be varied. One to three vaccinations administered at intervals of one to three weeks are usually effective.

The following examples are intended to further illustrate the invention without limiting its scope. The techniques used to infect and transfect cells, plaque purify virus, perform immunoblot analysis are widely practiced in the art.

EXAMPLE 1

GENERATION OF RECOMBINANT RACCOON POX VIRUSES EXPRESSING FIPV N AND E1 GENES

1. Cloning of FIPV N and E1 Genes and Preparation of Transfer Plasmids

The sequences of the E1 SEQ. ID. NO. 1 and N SEQ. ID. NO. 2 genes used in the present invention are shown in FIGS. 1A to 1E and 2A to 2G, respectively, of the specification. The methods for cloning of the N and E1 genes of FIPV and their insertion into a pSC11 transfer vector are detailed in European Patent Application 0,376,744, which is incorporated by reference. The plasmid used to clone the cDNA for the E1 and N genes is shown in FIGS. 3A and 3B. The pSC11 plasmids carrying the E1 and N genes are shown in FIGS. 4B and 4 B, respectively. The sequences of these plasmids are shown in FIGS. 5A to 5F (SEQ ID NO: 3) and FIGS. 6A to 6F (SEQ ID NO: 4).

To construct a pSC11 transfer plasmid containing both N and E1 genes, a 1.0 kb DNA fragment containing the vaccinia 7.5 promoter and the E1 gene was inserted downstream of the N gene in pSC11-FIPV N. The resulting plasmid was designated pSC11-FIPV N/E1.

2. Preparation of Recombinant Raccoon Poxviruses (RRPVs)

Monolayers of Vero cells (ATCC CCL 81) that were 80% confluent (approximately 5.times.10.sup.6 cells/100 mm tissue culture dish) were infected for 30-60 minutes at 37.degree. C. with wild-type raccoon pox virus (ATCC VR-338) at a multiplicity of infection MOI) of 0.1 TCID.sub.50 /cell. The medium (2 ml) consisted of Eagle's Minimum Essential Medium ("MEM", Gibco BRL #410-1500) containing 0.05% lactalbumin hydrolysate and 15 .mu.g/ml gentamicin sulfate and adjusted to pH 7.2 with sodium bicarbonate. After infection, the medium was removed and the cells were transfected with the pSC11-FIPV N, pSC11-FIPV E1, or pSC11 N/E1 transfer plasmid by cationic liposome-mediated transfection using Dioctadecylamidoglycyclsperimine-4-trifluoroacetic acid, (TRANSFECTAM) (Promega Corporation, Madison, Wis.) and dioleoyloxy)propyl-N,N,N-trimethylammonium methyl sulfate (DOTAP) (Boehringer Mannheim, Indianapolis, Ind.), respectively, per manufacturer's instructions. The cells were incubated with the DNA-liposomes mixture in 3 ml of MEM containing 5% fetal bovine serum (FBS) overnight at 37.degree. C. (5% CO.sub.2), after which the medium was replaced with 8 ml of fresh MEM-5% FBS. The transfected cells were incubated at 37.degree. C. (5% CO.sub.2) until greater than 80% showed cytopathic effects (CPE), which took approximately 3-4 days. The virus-cell lysates were then removed from the plates and subjected to two cycles of freeze-thawing before storage at -70.degree. C.

3. Isolation of Recombinant Raccoon Pox Virus Carrying the FIPV N Gene

RRPVs carrying the FIPV N gene (RRPV-FIPV N) were isolated and purified from the pSC11 -FIPV N Vero cell transfection by standard viral plaque purification methods. Monolayers of Vero cells (50-80% confluent) were infected with 2 ml of ten-fold serial dilutions (10.sup.-1 to 10.sup.-3) of the viral-cell lysates for 1 hour at 37.degree. C. After incubation, the media was removed and the infected cells were overlaid with 8-10 ml of 1.25% Noble agar containing MEM/5% FBS. The infected cells were then incubated for 3-4 days at 37.degree. C. (5% CO.sub.2), and overlaid again with 4 ml of 1.25% Nobel agar containing 0.5 X PBS and 600 .mu.g/ml 5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside (X-gal, States Biochemical Cleveland, Ohio). The plates were incubated at 37.degree. C. (5% CO.sub.2) for 4-16 hours, until blue (i.e. .beta.-galactosidase positive) viral plaques were observed. The recombinant viral plaques were picked with sterile blunt needles attached to a 1 cc syringe, suspended in 0.5 ml of 0.25 .mu.g/ml trypsin, vortexed vigorously, and incubated at 37.degree. C. for 15-30 min. The disrupted viral plaques were then inoculated onto 5.times.10.sup.5 Vero cells in 25 cm.sup.2 flasks and incubated at 37.degree. C. (5% CO.sub.2) until greater than 80% CPE was observed. The viral-cell lysates containing RRPV-FIPV N were subjected to two cycles of freeze-thawing and stored at -70.degree. C. Six individual RRPV-FIPV N clones were selected and plaque-purified five times as described above.

Isolation of Recombinant Raccoon Pox Virus Containing the FIPV E1 Gene

RRPVs carrying the FIPV E1 gene (RRPV-FIPV E1) were isolated and purified from pSC11-FIPV E1-transfected Vero cells using the methods described for rRPV-FIPV N, with some modifications. In this case, thymidine kinase deficient (tk-) RRPVs from the initial virus-cell lysates were selected on tk-RAT-2 cells (ATCC CRL 1764). This was performed by inoculating 1 ml of the initial virus-cell lysate onto a monolayer of RAT-2 cells in a 75 cm.sup.2 flask (approximately 5.times.10.sup.6 cells) in the presence of 5-bromodeoxyuridine (BrdU) at 30 .mu.g/ml in MEM. The infected monolayer was incubated at 37.degree. C. (5% CO.sub.2) for 3-4 days until greater than 70% CPE was observed. The tk-virus-cell lysates were subjected to two cycles of freeze-thawing before storage at -70.degree. C. Two individual RRPV-FIPV E1 clones were selected and subjected to six cycles of plaque purification as described above for RRPV-FIPV N.

5. Confirmation of FIPV N and E1 Genes in RRPV by Polymerase Chain Reaction

The presence of the FIPV N and E1 genes in the RRPVs was confirmed using the polymerase chain reaction (PCR). 90 .mu.l of a virus-cell lysate were incubated with 10 .mu.l of tenfold concentrated PCR lysis buffer (100 mM Tris-HCL buffer, pH 8.5; 500 mM KCl; 25 mM MgCl.sub.2 ; 5% Tween 20 (polyoxyethylenesorbitan monolaurate); 3 mg/ml Proteinase K) for 16 hours at 50.degree. C., then boiled for 10 min. 10 .mu.l of this lysate was used in the PCR. PCR was performed in 100 .mu.l of 10 mM Tris-HCL buffer, pH 8.3; 50 mM KCl; 200 uM of each deoxyribonucleotide triphosphate, 1.5 mM MgCl.sub.2 ; 30 pmoles of each oligonucleotide primer; and 2.5 units of AMPLITAQ DNA polymerase (Taq DNA polymerase) (Perkin-Elmer Cetus, Norwalk, Conn.). The primers used in the PCR for FIPV N were:

corresponding to nucleotides 68-91 and 721-744 of the FIPV N open reading frame (SEQ ID NO: 2) (primers 1 and 2, respectively). The primers used in the PCR for FIPV E1 were:

corresponding to nucleotides 334-355 and 721-742 of the FIPV E1 open reading frame SEQ. ID. NO. 1 (primers 3 and 4, respectively). The PCR amplifications were performed in a DNA Thermal Cycler Perkin-Elmer Cetus) by first heating the reaction mixes to 94.degree. C. for denaturation, and then performing 35 cycles of amplification, each consisting of 1 min at 95.degree. C., 1 min at 55.degree. C., 2 min at 72.degree. C., and, on the last cycle, a final incubation of 8 min at 72.degree. C. 10 .mu.l of the PCR products were resolved by electrophoresis in a horizontal-submarine 4% NUSIEVE (agarose) gel (FMC BioProducts, Rockland, Me.) in TAE buffer (40 mM Tris base, 20 mM sodium acetate, 1 mM EDTA, pH 7.2) by applying 5 V/cm for 1-2 hours. The DNA products were visualized by staining the gels with ethidium bromide.

PCR amplifications with the FIPV N and E1 primers gave DNA fragments of 676 and 408 nucleotides, respectively (FIG. 7). PCR amplifications using the pSC11 FIPV N and E1 transfer plasmids served as positive controls, and showed products of the predicted sizes. PCR amplifications using wild-type raccoon pox virus-Vero cell lysates served as a negative control, and no PCR products were observed in those samples.

Confirmation of RRPV FIPV N and E1 Protein Expression by Immunoblot Analysis

Confluent monolayers of Vero cells in a 25 cm.sup.2 flask (1-2.times.10.sup.6 cells) were infected with clones of either RRPV-FIPV N or RRPV-FIPV E1 at an MOI of 0.1. The infected cell were incubated at 37.degree. C. (5% CO.sub.2) for 2-3 days until approximately 80% of the cells showed cytopathic effects. A virus-cell lysate was prepared, and 20 .mu.l of the sample were added to 5 .mu.l of 5 X Laemmli sample buffer (0.3M Tris-HCI buffer, pH 6.8, containing 5% SDS, 50% glycerol, 0.4% bromophenol blue, and 3% 2-.beta.-mercaptoethanol) and heated at 95.degree. C. for 5 min. The denatured protein samples were separated By SDS/polyacrylamide electrophoresis using a 4-15% gradient polyacrylamide gel as described previously. Maniatis et al., Molecular Cloning: A Laboratory Manual, 1982, Cold Spring Harbor Press. After electrophoresis, the proteins were transferred to nitrocellulose (Bio-Rad Laboratories, Hercules, Calif.) by electrotransfer using a Bio-Rad transfer apparatus per manufacturer's instructions. The transfer was performed in 25 mM Tris-HCI buffer, containing 0.2M glycine and 20% methanol, for 45 minutes at 50 V with constant current.

FIPV N and E1 proteins were visualized on the nitrocellulose filter using specific antibodies. Davis et al., Basic Methods in Molecular Biology, 1986, Elsevier Science Publishing Company, New York, N.Y. The filter was rinsed in phosphate buffered saline pH 7.4 containing 0.1% Tween-20 (polyoxyethylenesorbitan monolaurate) ("PBS-TW"), after which non-specific sites were blocked by overnight incubation at 4.degree. C. in PBS containing 1% bovine serum albumin (PBS-BSA) followed by a 15 min wash in PBS-TW. The filter was then incubated for 30 min at room temperature with anti-FIPV antibodies, which consisted of ascites fluid from a FIPV (strain 79-1146)-infected cat, diluted 1:100 in PBS-TW containing 1% BSA ("PBS-TW-BSA"). After four 5 min washes in PBS-TW, the filter was incubated for 30 min at room temperature with a secondary antibody consisting of biotin-labeled mouse anti-cat IgG antibody (Kirkegaard & Perry Laboratories Inc., Gaithersburg, Md.) that had been diluted 1:2000 in PBS-TW-BSA, followed by four 5 min washes in PBS-TW. The filter was then incubated for 30 min at room temperature with horseradish peroxidase-conjugated streptavidin (Kirkegaard a Perry Laboratories Inc.) that had been diluted 1:1000 in PBS-TW. After the filter was washed four times (5 min each) in PBS-TW, the antigen-antibody complexes were visualized with peroxidase chromogenic substrate (Kirkegaard & Perry Laboratories Inc.). Sucrose-gradient purified FIPV and wild-type raccoon pox virus-Vero cell lysates were used as the positive and negative controls, respectively. A typical immunoblot is shown in FIG. 8.

7. Raccoon Poxvirus Titration

Serial tenfold dilutions of virus are prepared in MEM and inoculated in replicates of five onto Vero cells (1.times.10.sup.4 cells per well) in a 96 well plate. Virus preparations may be pretreated by dilution into an equal volume of 0.5% trypsin and incubation at 37.degree. C. for 30 min in order to release virus from inclusions. Plates are incubated for 3-5 days at 37.degree. C. (5% CO.sub.2) and observed for cytopathology typical of raccoon poxvirus. Titers are calculated as 50% endpoints based on cytopathology using the methods of Reed and Muench, The American Journal of Hygiene 27(3):493-497) (1938).

EXAMPLE 2

PREPARATION OF VACCINE AND TESTING FOR EFFICACY IN CATS

1. Preparation of Master Seeds of RRPV-FIPV N and E1 Viruses

A single clone of each recombinant virus was selected for large-scale expansion to serve as a master seed virus. The criteria for selection were: 1) Demonstration of purity. Polymerase chain reaction was utilized to insure that the clone was uncontaminated with wild type virus. 2) Demonstration of adequate recombinant proten expression by Western blot or other antigen detection methods.

All recombinant virus expansions and titrations were done on Vero cells in MEM containing 2.5% FBS. Each plaque purified virus clone was expanded by inoculating a confluent monolayer of Vero cells in a 150 cm.sup.2 flask (1.times.10.sup.7 cells) with 1 ml of viral-cell lysate (approximately 10.sup.7 infectious virus particles), and incubating at 37.degree. C. (5% CO.sub.2) until 100% cytopathic effect was observed (2-3 days). This virus-cell lysate was titrated on Vero cells as described in Example 1, and served as a premaster seed virus stock to obtain the master seed virus. The MOI to be used to produce the highest titer master seed virus was determined by inoculating a confluent monolayer of Vero cells in a roller bottle (1.times.10.sup.8 cells) with various MOIs of recombinant virus (e.g. 0.1, 0.05, 0.01, 0.005, and 0.001 TCID.sub.50 /cell.) The infected cells were incubated at 37.degree. C. until greater than 80% CPE was observed (approximately 3 days), and the titers of each infected roller bottle was determined. The master seed viruses were aliquoted into 1.5 ml ampules, which were sealed and stored in a liquid nitrogen freezer.

2. Preparation of Vaccines

3.times.10.sup.7 Vero cells were seeded into 850 cm.sup.2 roller bottles in 200 ml of growth media (MEM containing 0.5% lactalbumin hydrolysate and 5% FBS) and incubated for 18 hours at 37.degree. C. The next day, the medium was removed from the cells and replaced with 50ml of RRPV-FIPV N virus diluted to an MOI of 0.01 in infection media (MEM containing 0.5% lactalbumin hydrolysate and 2.5% FBS). The virus used was at the second passage beyond the master seed preparation. Virus was allowed to absorb to the cells for 30 min at 37.degree. C., after which the volume of medium was adjusted to 150 ml per roller bottle. Roller bottles were incubated at 37.degree. C. until 100% cytopathology was evident (3 days). The virus-cell lysate was harvested and stored frozen (-70.degree. C.). The virus titer was determined to be 10.sup.6.97 TCID.sub. /ml.

RRPV-FIPV E1 stocks were prepared in the same manner, except that an MOI of 0.1 was used. The final virus preparation was titered and found to contain 10.sup.6.5 TCID.sub.50 /ml. Wild type raccoon poxvirus was grown using the same methods as described above, and contained 10.sup.6.44 TCID.sub.50 /ml.

3. Vaccination

A group of twenty-four 9-month-old cats (specific pathogen-free, Harlan Sprague Dawley, Madison, Wis.), comprising seven males and seventeen females, was used to demonstrate the efficacy of the RRPV-FIPV N vaccine. Cats were divided into five groups and vaccinated twice, 21 days apart, as indicated below:

______________________________________ # Volume Viral Dose Vaccinaton Group Cats Vaccine (ml) (TCID.sub.50) Route* ______________________________________ 1 5 RRPV-FIPV N 3 10.sup.7.44 SC 2 5 RRPV-FIPV N 1 10.sup.6.97 IM 3 5 RRPV-FIPV N 3 10.sup.7.44 ORAL 4 4 RRPV-FIPV N 3 10.sup.6.44 SC (1:10 Dilution) 5 5 Wild Type REV 3 10.sup.6.44 SC ______________________________________ *SC = Subcutaneous IM = Intramuscular Oral = Oral

4. Challenge

Two weeks following the second vaccination, cats were orally inoculated with 10.sup.3.4 TCID.sub.50 of Feline Enteric Coronavirus (strain 79-1683, ATCC VR-989). This virus induces a subclinical infection which can enhance subsequent FIPV infection. Three weeks later, cats were orally challenged with 10.sup.3.4 TCID.sub.50 of FIPV (strain 79-1146, ATCC VR-990). Cats were monitored weekly for a total of 64 days after challenge for signs of clinical disease including: fever, icterus, leukopenia, anemia, weight loss, anorexia, depression, dehydration, and peritoneal swelling. Cats deemed moribund were euthanized by the attending veterinarian and post-mortem pathological examination was performed. Clinical disease signs were scored as follows:

______________________________________ SIGN SCORE ______________________________________ Fever 103.0-10.39.degree. F. 1 point/day* 104.0-104.9.degree. F. 2 points/day .gtoreq.105.0.degree. F. 3 points/day Dehydration 1 point/day Depression 1 point/day Anorexia 1 point/day Peritoneal Swelling 1 point/day Icterus 1 point/day Weight Loss >20% 1 point per observation >30% 2 points per observation >50% 5 points per observation Leukopenia decrease of 50% 3 points per observation counts <6000 2 points per observation Hematocrit 3 points per observation <25% PCV Death 25 points ______________________________________ *For cats with baseline temperatures averaging 103.degree. F., no points will be scored until temperatures are in excess of 1.degree. F. above baseline.

5. Evaluation of Induced Immunity to FIPV

Inoculation with virulent FIPV induced a fatal infection in 4/5 (80%) of the control cats, which were vaccinated with wild type raccoon poxvirus (Table 1). Both effusive and non-effusive forms of the disease were noted in the control cats. On the other hand, clinical disease was essentially absent after challenge of the subcutaneous vaccinates. The sporadic fever in these cats could be attributed to excitability and the slight anemia on one day in cat 1264 is not a significant finding. The subcutaneous vaccinates showed a statistically significant reduction in clinical signs (p<0.05, by ANOVA) and death (p<0.01, by Chi Square Analysis) when compared to the control cats.

The intramuscular route of vaccination was less effective in that 2/5 (40%) of the cats succumbed to FIPV-induced disease. However, the onset of disease in these cats was delayed when compared to the controls. The decreased efficacy may be related to the lower titer of virus inoculated into these cats because only a 1 ml dose could be administered by this route. There was also decreased efficacy when cats were inoculated by the oral route (60% mortality) which may indicate the need for a higher virus dose when vaccinated by this route.

The protection conferred against FIPV-caused disease by the subcutaneously administered vaccine was shown to be dose-dependent, confirming the benefit of a high-titer RRPV-FIPV vaccine in inducing protection against clinical disease induced by FIPV virus. A suitable vaccine dose contains viral antigen in the range of 10.sup.4 -10.sup.8 TCID.sub.50 /ml, preferably 10.sup.7 -10.sup.8 TCID.sub.50 /ml. A typical dose for administration to cats is 1-3 ml, and delivery by the subcutaneous route is preferred.

TABLE 1 __________________________________________________________________________ TOTAL CLINICAL SCORES FOLLOWING CHALLENGE WITH FIPV CAT ID Fever Weight Loss Leukopenia Anemia Chemical Signs Death* Total Score __________________________________________________________________________ SUBCUTANEOUS VACCINATES 1260 0 0 0 0 0 0 0 1262 1 0 0 0 0 0 1 1264 1 0 0 0 0 0 4 1266 0 0 0 0 0 0 0 1268 2 0 0 0 0 0 2 INTRAMUSCULUAR VACCINATES 1270 13 6 0 0 33 25 77 1272 2 0 0 0 0 0 2 1297 23 0 0 0 34 25 91 1301 3 0 0 0 0 0 3 ORAL VACCINATES 1303 1 0 4 0 0 0 5 1305 0 1 0 0 5 0 6 1307 3 1 0 0 4 25 33 1309 2 1 6 0 6 25 40 1311 4 3 0 0 18 25 50 1/10 DOSE SUBCUTANEOUS VACCINATES 1313 29 5 9 3 36 25 107 1315 17 0 0 0 20 25 62 1317 3 0 3 0 9 25 40 1319 1 0 0 0 6 0 7 CONTROLS 1321 39 3 18 3 63 25 151 1323 5 0 2 0 14 25 46 1327 0 1 2 0 15 25 43 1329 5 1 6 0 11 25 48 1337 1 0 0 0 0 0 1 __________________________________________________________________________

__________________________________________________________________________ SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 10 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 789 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Feline infectious peritonitis virus (vii) IMMEDIATE SOURCE: (B) CLONE: FIPV E1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: ATGAAGTACATTTTGCTAATACTCGCGTGCATAATTGCATGCGTTTATGGTGAACGCTAC60 TGTGCCATGCAAGACAGTGGCTTGCAGTGTATTAATGGCACAAATTCAAGATGTCAAACC120 TGCTTTGAACGTGGTGATCTTATTTGGCATCTTGCTAACTGGAACTTCAGCTGGTCTGTA180 ATATTGATTGTTTTTATAACAGTGTTACAATATGGCAGACCACAATTTAGCTGGCTCGTT240 TATGGCATTAAAATGCTGATCATGTGGCTATTATGGCCTATTGTTCTAGCGCTTACGATT300 TTTAATGCATACTCTGAGTACCAAGTTTCCAGATATGTAATGTTCGGCTTTAGTGTTGCA360 GGTGCAGTTGTAACGTTTGCACTTTGGATGATGTATTTTGTGAGATCTGTTCAGCTATAT420 AGAAGAACCAAATCATGGTGGTCTTTTAATCCTGAGACTAATGCAATTCTTTGTGTTAAT480 GCATTGGGTAGAAGTTATGTGCTTCCCTTAGATGGTACTCCTACAGGTGTTACCCTTACT540 CTACTTTCAGGAAATCTATATGCTGAAGGTTTCAAAATGGCTGGTGGTTTAACCATCGAG600 CATTTGCCTAAATACGTCATGATTGCTACACCTAGTAGAACCATCGTTTATACATTAGTT660 GGAAAACAATTAAAAGCAACTACTGCCACAGGATGGGCTTACTACGTAAAATCTAAAGCT720 GGTGATTACTCAACAGAAGCACGTACTGACAATTTGAGTGAACATGAAAAATTATTACAT780 ATGGTGTAA789 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1134 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Feline infectious peritonitis virus (vii) IMMEDIATE SOURCE: (B) CLONE: FIPV N (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: ATGGCCACACAGGGACAACGCGTCAACTGGGGAGATGAACCTTCCAAAAGACGTGGTCGT60 TCTAACTCTCGTGGTCGGAAGAATAATGATATACCTTTGTCATTCTACAACCCCATTACC120 CTCGAACAAGGATCTAAATTTTGGAATTTATGTCCGAGAGACCTTGTTCCCAAAGGAATA180 GGTAATAAGGATCAACAAATTGGTTATTGGAATAGACAGATTCGTTATCGTATTGTAAAA240 GGCCAGCGTAAGGAACTCGCTGAGAGGTGGTTCTTTTACTTCTTAGGTACAGGACCTCAT300 GCTGATGCTAAATTCAAAGACAAGATTGATGGAGTCTTCTGGGTTGCAAGGGATGGTGCC360 ATGAACAAGCCCACAACGCTTGGCACTCGTGGAACCAATAACGAATCCAAACCACTGAGA420 TTTGATGGTAAGATACCGCCACAGTTTCAGCTTGAAGTGAACCGTTCTAGGAACAATTCA480 AGGTCTGGTTCTCAGTCTAGATCTGTTTCAAGAAACAGATCTCAATCTAGAGGAAGACAC540 CATTCCAATAACCAGAATAATAATGTTGAGGATACAATTGTAGCCGTGCTTGAAAAATTA600 GGTGTTACTGACAAACAAAGGTCACGTTCTAAACCTAGAGAACGTAGTGATTCCAAACCT660 AGGGACACAACACCTAAGAATGCCAACAAACACACCTGGAAGAAAACTGCAGGCAAGGGA720 GATGTGACAACTTTCTATGGTGCTAGAAGTAGTTCAGCTAACTTTGGTGATAGTGATCTC780 GTTGCCAATGGTAACGCTGCCAAATGCTACCCTCAGATAGCTGAATGTGTTCCATCAGTG840 TCTAGCATAATCTTTGGCAGTCAATGGTCTGCTGAAGAAGCTGGTGATCAAGTGAAAGTC900 ACGCTCACTCACACCTACTACCTGCCAAAGGATGATGCCAAAACTAGTCAATTCCTAGAA960 CAGATTGACGCTTACAAGCGACCTTCTGAAGTGGCTAAGGATCAGAGGCAAAGAAGATCC1020 CGTTCTAAGTCTGCTGATAAGAAGCCTGAGGAGTTGTCTGTAACTCTTGTGGAGGCATAC1080 ACAGATGTGTTTGATGACACACAGGTTGAGATGATTGATGAGGTTACGAACTAA1134 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8710 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Feline infectious peritonitis virus (vii) IMMEDIATE SOURCE: (B) CLONE: psc11f1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCT60 TAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTC120 TAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAA180 TATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTT240 GCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCT300 GAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATC360 CTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTA420 TGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACAC480 TATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGC540 ATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAAC600 TTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGG660 GATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGAC720 GAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGC780 GAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTT840 GCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGA900 GCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCC960 CGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAG1020 ATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCA1080 TATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATC1140 CTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCA1200 GACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGC1260 TGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTA1320 CCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTT1380 CTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTC1440 GCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGG1500 TTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCG1560 TGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAG1620 CATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGC1680 AGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTAT1740 AGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGG1800 GGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGC1860 TGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATT1920 ACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCA1980 GTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCG2040 ATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAAC2100 GCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCG2160 GCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGAC2220 CATGATTACGCCAAGCTTTTGCGATCAATAAATGGATCACAACCAGTATCTCTTAACGAT2280 GTTCTTCGCAGATGATGATTCATTTTTTAAGTATTTGGCTAGTCAAGATGATGAAATCTT2340 CATTATCTGATATATTGCAAATCACTCAATATCTAGACTTTCTGTTATTATTATTGATCC2400 AATCAAAAAATAAATTAGAAGCCGTGGGTCATTGTTATGAATCTCTTTCAGAGGAATACA2460 GACAATTGACAAAATTCACAGACTTTCAAGATTTTAAAAAACTGTTTAACAAGGTCCCTA2520 TTGTTACAGATGGAAGGGTCAAACTTAATAAAGGATATTTGTTCGACTTTGTGATTAGTT2580 TGATGCGATTCAAAAAAGAATCCTCTCTAGCTACCACCGCAATAGATCCTGTTAGATACA2640 TAGATCCTCGTCGCAATATCGCATTTTCTAACGTGATGGATATATTAAAGTCGAATAAAG2700 TGAACAATAATTAATTCTTTATTGTCATCATGAACGGCGGACATATTCAGTTGATAATCG2760 GCCCCATGTTTTCAGGTAAAAGTACAGAATTAATTAGACGAGTTAGACGTTATCAAATAG2820 CTCAATATAAATGCGTGACTATAAAATATTCTAACGATAATAGATACGGAACGGGACTAT2880 GGACGCATGATAAGAATAATTTTGAAGCATTGGAAGCAACTAAACTATGTGATCTCTTGG2940 AATCAATTACAGATTTCTCCGTGATAGGTATCGATGAAGGACAGTTCTTTCCAGACATTG3000 TTGAATTCCGAGCTTGGCTGCAGGTCGGGGATCCCCCCTGCCCGGTTATTATTATTTTTG3060 ACACCAGACCAACTGGTAATGGTAGCGAACGGCGCTCAGCTGAATTCCGCCGATACTGAC3120 GGGCTCCAGGAGTCGTCGCCACCAATCCCCATATGGAAACCGTCGATATTCAGCCATGTG3180 CCTTCTTCCGCGTGCAGCAGATGGCGATGGCTGGTTTCCATCAGTTGCTGTTGACTGTAG3240 CGGCTGATGTTGAACTGGAAGTCGCCGCGCCACTGGTGTGGGCCATAATTCAATTCGCGC3300 GTCCCGCAGCGCAGACCGTTTTCGCTCGGGAAGACGTACGGGGTATACATGTCTGACAAT3360 GGCAGATCCCAGCGGTCAAAACAGGCGGCAGTAAGGCGGTCGGGATAGTTTTCTTGCGGC3420 CCTAATCCGAGCCAGTTTACCCGCTCTGCTACCTGCGCCAGCTGGCAGTTCAGGCCAATC3480 CGCGCCGGATGCGGTGTATCGCTCGCCACTTCAACATCAACGGTAATCGCCATTTGACCA3540 CTACCATCAATCCGGTAGGTTTTCCGGCTGATAAATAAGGTTTTCCCCTGATGCTGCCAC3600 GCGTGACCGGTCGTAATCAGCACCGCATCAGCAAGTGTATCTGCCGTGCACTGCAACAAC3660 GCTGCTTCGGCCTGGTAATGGCCCGCCGCCTTCCAGCGTTCGACCCAGGCGTTAGGGTCA3720 ATGCGGGTCGCTTCACTTACGCCAATGTCGTTATCCAGCGGTGCACGGGTGAACTGATCG3780 CGCAGCGGCGTCAGCAGTTGTTTTTTATCGCCAATCCACATCTGTGAAAGAAAGCCTGAC3840 TGGCGGTTAAATTGCCAACGCTTATTACCCAGCTCGATGCAAAAATCCATTTCGCTGGTG3900 GTCAGATGCGGGATGGCGTGGGACGCGGCGGGGAGCGTCACACTGAGGTTTTCCGCCAGA3960 CGCCACTGCTGCCAGGCGCTGATGTGCCCGGCTTCTGACCATGCGGTCGCGTTCGGTTGC4020 ACTACGCGTACTGTGAGCCAGAGTTGCCCGGCGCTCTCCGGCTGCGGTAGTTCAGGCAGT4080 TCAATCAACTGTTTACCTTGTGGAGCGACATCCAGAGGCACTTCACCGCTTGCCAGCGGC4140 TTACCATCCAGCGCCACCATCCAGTGCAGGAGCTCGTTATCGCTATGACGGAACAGGTAT4200 TCGCTGGTCACTTCGATGGTTTGCCCGGATAAACGGAACTGGAAAAACTGCTGCTGGTGT4260 TTTGCTTCCGTCAGCGCTGGATGCGGCGTGCGGTCGGCAAAGACCAGACCGTTCATACAG4320 AACTGGCGATCGTTCGGCGTATCGCCAAAATCACCGCCGTAAGCCGACCACGGGTTGCCG4380 TTTTCATCATATTTAATCAGCGACTGATCCACCCAGTCCCAGACGAAGCCGCCCTGTAAA4440 CGGGGATACTGACGAAACGCCTGCCAGTATTTAGCGAAACCGCCAAGACTGTTACCCATC4500 GCGTGGGCGTATTCGCAAAGGATCAGCGGGCGCGTCTCTCCAGGTAGCGAAAGCCATTTT4560 TTGATGGACCATTTCGGCACAGCCGGGAAGGGCTGGTCTTCATCCACGCGCGCGTACATC4620 GGGCAAATAATATCGGTGGCCGTGGTGTCGGCTCCGCCGCCTTCATACTGCACCGGGCGG4680 GAAGGATCGACAGATTTGATCCAGCGATACAGCGCGTCGTGATTAGCGCCGTGGCCTGAT4740 TCATTCCCCAGCGACCAGATGATCACACTCGGGTGATTACGATCGCGCTGCACCATTCGC4800 GTTACGCGTTCGCTCATCGCCGGTAGCCAGCGCGGATCATCGGTCAGACGATTGATTGGC4860 ACCATGCCGTGGGTTTCAATATTGGCTTCATCCACCACATACAGGCCGTAGCGGTCGCAC4920 AGCGTGTACCACAGCGGATGGTTCGGATAATGCGAACAGCGCACGGCGTTAAAGTTGTTC4980 TGCTTCATCAGCAGGATATCCTGCACCATCGTCTGCTCATCCATGACCTGACCATGCAGA5040 GGATGATGCTCGTGACGGTTAACGCCTCGAATCAGCAACGGCTTGCCGTTCAGCAGCAGC5100 AGACCATTTTCAATCCGCACCTCGCGGAAACCGACATCGCAGGCTTCTGCTTCAATCAGC5160 GTGCCGTCGGCGGTGTGCAGTTCAACCACCGCACGATAGAGATTCGGGATTTCGGCGCTC5220 CACAGTTTCGGGTTTTCGACCTTGAGACGTAGTGTGACGCGATCGGCATAACCACCACGC5280 TCATCGATAATTTCACCGCCGAAAGGCGCGGTGCCGCTGGCGACCTGCGTTTCACCCTGC5340 CATAAAGAAACTGTTACCCGTAGGTAGTCACGCAACTCGCCGCACATCTGAACTTCAGCC5400 TCCAGTACAGCGCGGCTGAAATCATCATTAAAGCGAGTGGCAACATGGAAATCGCTGATT5460 TGTGTAGTCGGTTTATGCAGCAACGAGACGTCACGGAAAATGCCGCTCATCCGCCACATA5520 TCCTGATCTTCCAGATAACTGCCGTCACTCCAACGCAGCACCATCACCGCGAGGCGGTTT5580 TCTCCGGCGCGTAAAAATGCGCTCAGGTCAAATTCAGACGGCAAACGACTGTCCTGGCCG5640 TAACCGACCCAGCGCCCGTTGCACCACAGATGAAACGCCGAGTTAACGCCATCAAAAATA5700 ATTCGCGTCTGGCCTTCCTGTAGCCAGCTTTCATCAACATTAAATGTGAGCGAGTAACAA5760 CCCGTCGGATTCTCCGTGGGAACAAACGGCGGATTGACCGTAATGGGATAGGTTACGTTG5820 GTGTAGATGGGCGCATCGTAACCGTGCATCTGCCAGTTTGAGGGGACGACGACAGTATCG5880 GCCTCAGGAAGATCGCACTCCAGCCAGCTTTCCGGCACCGCTTCTGGTGCCGGAAACCAG5940 GCAAAGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCC6000 TCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTA6060 ACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGGATCCCTCGAGGAATTCATT6120 TATAGCATAGAAAAAAACAAAATGAAATTCTACTATATTTTTACATACATATATTCTAAA6180 TATGAAAGTGGTGATTGTGACTAGCGTAGCATCGCTTCTAGACATATACTATATAGTAAT6240 ACCAATACTCAAGACTACGAAACTGATACAATCTCTTATCATGTGGGTAATGTTCTCGAT6300 GTCGAATAGCCATATGCCGGTAGTTGCGATATACATAAACTGATCACTAATTCCAAACCC6360 ACCCGCTTTTTATAGTAAGTTTTTCACCCATAAATAATAAATACAATAATTAATTTCTCG6420 TAAAAGTAGAAAATATATTCTAATTTATTGCACGGTAAGGAAGTAGAATCATAAAGAACA6480 GTGACGGATCCCAATTCGGGCATTTTTGGTTTGAACTAAACAAAATGAAGTACATTTTGC6540 TAATACTCGCGTGCATAATTGCATGCGTTTATGGTGAACGCTACTGTGCCATGCAAGACA6600 GTGGCTTGCAGTGTATTAATGGCACAAATTCAAGATGTCAAACCTGCTTTGAACGTGGTG6660 ATCTTATTTGGCATCTTGCTAACTGGAACTTCAGCTGGTCTGTAATATTGATTGTTTTTA6720 TAACAGTGTTACAATATGGCAGACCACAATTTAGCTGGCTCGTTTATGGCATTAAAATGC6780 TGATCATGTGGCTATTATGGCCTATTGTTCTAGCGCTTACGATTTTTAATGCATACTCTG6840 AGTACCAAGTTTCCAGATATGTAATGTTCGGCTTTAGTGTTGCAGGTGCAGTTGTAACGT6900 TTGCACTTTGGATGATGTATTTTGTGAGATCTGTTCAGCTATATAGAAGAACCAAATCAT6960 GGTGGTCTTTTAATCCTGAGACTAATGCAATTCTTTGTGTTAATGCATTGGGTAGAAGTT7020 ATGTGCTTCCCTTAGATGGTACTCCTACAGGTGTTACCCTTACTCTACTTTCAGGAAATC7080 TATATGCTGAAGGTTTCAAAATGGCTGGTGGTTTAACCATCGAGCATTTGCCTAAATACG7140 TCATGATTGCTACACCTAGTAGAACCATCGTTTATACATTAGTTGGAAAACAATTAAAAG7200 CAACTACTGCCACAGGATGGGCTTACTACGTAAAATCTAAAGCTGGTGATTACTCAACAG7260 AAGCACGTACTGACAATTTGAGTGAACATGAAAAATTATTACATATGGTGTAACTAAACT7320 TTCAAATGGGGGAATTCTGTGAGCGTATGGCAAACGAAGGAAAAATTAGTTATAGTAGCC7380 GCACTCGATGGGACATTTCAACGTAAACCGTTTAATAATATTTTGAATCTTATTCCATTA7440 TCTGAAATGGTGGTAAAACTAACTGCTGTGTGTATGAAATGCTTTAAGGAGGCTTCCTTT7500 TCTAAACGATTGGGTGAGGAAACCGAGATAGAAATAATAGGAGGTAATGATATGTATCAA7560 TCGGTGTGTAGAAAGTGTTACATCGACTCATAATATTATATTTTTTATCTAAAAAACTAA7620 AAATAAACATTGATTAAATTTTAATATAATACTTAAAAATGGATGTTGTGTCGTTAGATA7680 AACCGTTTATGTATTTTGAGGAAATTGATAATGAGTTAGATTACGAACCAGAAAGTGCAA7740 ATGAGGTCGCAAAAAAACTGCCGTATCAAGGACAGTTAAAACTATTACTAGGAGAATTAT7800 TTTTTCTTAGTAAGTTACAGCGACACGGTATATTAGATGGTGCCACCGTAGTGTATATAG7860 GATCTGCTCCCGGTACACATATACGTTATTTGAGAGATCATTTCTATAATTTAGGAGTGA7920 TCATCAAATGGATGCTAATTGACGGCCGCCATCATGATCCTATTTTAAATGGATTGCGTG7980 ATGTGACTCTAGTGACTCGGTTCGTTGATGAGGAATATCTACGATCCATCAAAAAACAAC8040 TGCATCCTTCTAAGATTATTTTAATTTCTGATGTGAGATCCAAACGAGGAGGAAATGAAC8100 CTAGTACGGCGGATTTACTAAGTAATTACGCTCTACAAAATGTCATGATTAGTATTTTAA8160 ACCCCGTGGCGTCTAGTCTTAAATGGAGATGCCCGTTTCCAGATCAATGGATCAAGGACT8220 TTTATATCCCACACGGTAATAAAATGTTACAACCTTTTGCTCCTTCATATTCAGGGCCGT8280 CGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGC8340 ACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCA8400 ACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCTTTACGCATCT8460 GTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACCATCTGCTCTGATGCCGCATA8520 GTTAAGCCAGTACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCC8580 GCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACA8640 AGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACG8700 CGCGAGGCAG8710 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9019 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Feline immunodeficiency virus (vii) IMMEDIATE SOURCE: (B) CLONE: psc11e1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTT60 AGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCT120 AAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAAT180 ATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTG240 CGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTG300 AAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCC360 TTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTAT420 GTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACT480 ATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCA540 TGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACT600 TACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGG660 ATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACG720 AGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCG780 AACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTG840 CAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAG900 CCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCC960 GTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGA1020 TCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCAT1080 ATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCC1140 TTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAG1200

ACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCT1260 GCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTAC1320 CAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTC1380 TAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCG1440 CTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGT1500 TGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGT1560 GCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGC1620 ATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCA1680 GGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATA1740 GTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGG1800 GGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCT1860 GGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTA1920 CCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAG1980 TGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGA2040 TTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACG2100 CAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGG2160 CTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACC2220 ATGATTACGCCAAGCTTTTGCGATCAATAAATGGATCACAACCAGTATCTCTTAACGATG2280 TTCTTCGCAGATGATGATTCATTTTTTAAGTATTTGGCTAGTCAAGATGATGAAATCTTC2340 ATTATCTGATATATTGCAAATCACTCAATATCTAGACTTTCTGTTATTATTATTGATCCA2400 ATCAAAAAATAAATTAGAAGCCGTGGGTCATTGTTATGAATCTCTTTCAGAGGAATACAG2460 ACAATTGACAAAATTCACAGACTTTCAAGATTTTAAAAAACTGTTTAACAAGGTCCCTAT2520 TGTTACAGATGGAAGGGTCAAACTTAATAAAGGATATTTGTTCGACTTTGTGATTAGTTT2580 GATGCGATTCAAAAAAGAATCCTCTCTAGCTACCACCGCAATAGATCCTGTTAGATACAT2640 AGATCCTCGTCGCAATATCGCATTTTCTAACGTGATGGATATATTAAAGTCGAATAAAGT2700 GAACAATAATTAATTCTTTATTGTCATCATGAACGGCGGACATATTCAGTTGATAATCGG2760 CCCCATGTTTTCAGGTAAAAGTACAGAATTAATTAGACGAGTTAGACGTTATCAAATAGC2820 TCAATATAAATGCGTGACTATAAAATATTCTAACGATAATAGATACGGAACGGGACTATG2880 GACGCATGATAAGAATAATTTTGAAGCATTGGAAGCAACTAAACTATGTGATCTCTTGGA2940 ATCAATTACAGATTTCTCCGTGATAGGTATCGATGAAGGACAGTTCTTTCCAGACATTGT3000 TGAATTCCGAGCTTGGCTGCAGGTCGGGGATCCCCCCTGCCCGGTTATTATTATTTTTGA3060 CACCAGACCAACTGGTAATGGTAGCGAACGGCGCTCAGCTGAATTCCGCCGATACTGACG3120 GGCTCCAGGAGTCGTCGCCACCAATCCCCATATGGAAACCGTCGATATTCAGCCATGTGC3180 CTTCTTCCGCGTGCAGCAGATGGCGATGGCTGGTTTCCATCAGTTGCTGTTGACTGTAGC3240 GGCTGATGTTGAACTGGAAGTCGCCGCGCCACTGGTGTGGGCCATAATTCAATTCGCGCG3300 TCCCGCAGCGCAGACCGTTTTCGCTCGGGAAGACGTACGGGGTATACATGTCTGACAATG3360 GCAGATCCCAGCGGTCAAAACAGGCGGCAGTAAGGCGGTCGGGATAGTTTTCTTGCGGCC3420 CTAATCCGAGCCAGTTTACCCGCTCTGCTACCTGCGCCAGCTGGCAGTTCAGGCCAATCC3480 GCGCCGGATGCGGTGTATCGCTCGCCACTTCAACATCAACGGTAATCGCCATTTGACCAC3540 TACCATCAATCCGGTAGGTTTTCCGGCTGATAAATAAGGTTTTCCCCTGATGCTGCCACG3600 CGTGACCGGTCGTAATCAGCACCGCATCAGCAAGTGTATCTGCCGTGCACTGCAACAACG3660 CTGCTTCGGCCTGGTAATGGCCCGCCGCCTTCCAGCGTTCGACCCAGGCGTTAGGGTCAA3720 TGCGGGTCGCTTCACTTACGCCAATGTCGTTATCCAGCGGTGCACGGGTGAACTGATCGC3780 GCAGCGGCGTCAGCAGTTGTTTTTTATCGCCAATCCACATCTGTGAAAGAAAGCCTGACT3840 GGCGGTTAAATTGCCAACGCTTATTACCCAGCTCGATGCAAAAATCCATTTCGCTGGTGG3900 TCAGATGCGGGATGGCGTGGGACGCGGCGGGGAGCGTCACACTGAGGTTTTCCGCCAGAC3960 GCCACTGCTGCCAGGCGCTGATGTGCCCGGCTTCTGACCATGCGGTCGCGTTCGGTTGCA4020 CTACGCGTACTGTGAGCCAGAGTTGCCCGGCGCTCTCCGGCTGCGGTAGTTCAGGCAGTT4080 CAATCAACTGTTTACCTTGTGGAGCGACATCCAGAGGCACTTCACCGCTTGCCAGCGGCT4140 TACCATCCAGCGCCACCATCCAGTGCAGGAGCTCGTTATCGCTATGACGGAACAGGTATT4200 CGCTGGTCACTTCGATGGTTTGCCCGGATAAACGGAACTGGAAAAACTGCTGCTGGTGTT4260 TTGCTTCCGTCAGCGCTGGATGCGGCGTGCGGTCGGCAAAGACCAGACCGTTCATACAGA4320 ACTGGCGATCGTTCGGCGTATCGCCAAAATCACCGCCGTAAGCCGACCACGGGTTGCCGT4380 TTTCATCATATTTAATCAGCGACTGATCCACCCAGTCCCAGACGAAGCCGCCCTGTAAAC4440 GGGGATACTGACGAAACGCCTGCCAGTATTTAGCGAAACCGCCAAGACTGTTACCCATCG4500 CGTGGGCGTATTCGCAAAGGATCAGCGGGCGCGTCTCTCCAGGTAGCGAAAGCCATTTTT4560 TGATGGACCATTTCGGCACAGCCGGGAAGGGCTGGTCTTCATCCACGCGCGCGTACATCG4620 GGCAAATAATATCGGTGGCCGTGGTGTCGGCTCCGCCGCCTTCATACTGCACCGGGCGGG4680 AAGGATCGACAGATTTGATCCAGCGATACAGCGCGTCGTGATTAGCGCCGTGGCCTGATT4740 CATTCCCCAGCGACCAGATGATCACACTCGGGTGATTACGATCGCGCTGCACCATTCGCG4800 TTACGCGTTCGCTCATCGCCGGTAGCCAGCGCGGATCATCGGTCAGACGATTGATTGGCA4860 CCATGCCGTGGGTTTCAATATTGGCTTCATCCACCACATACAGGCCGTAGCGGTCGCACA4920 GCGTGTACCACAGCGGATGGTTCGGATAATGCGAACAGCGCACGGCGTTAAAGTTGTTCT4980 GCTTCATCAGCAGGATATCCTGCACCATCGTCTGCTCATCCATGACCTGACCATGCAGAG5040 GATGATGCTCGTGACGGTTAACGCCTCGAATCAGCAACGGCTTGCCGTTCAGCAGCAGCA5100 GACCATTTTCAATCCGCACCTCGCGGAAACCGACATCGCAGGCTTCTGCTTCAATCAGCG5160 TGCCGTCGGCGGTGTGCAGTTCAACCACCGCACGATAGAGATTCGGGATTTCGGCGCTCC5220 ACAGTTTCGGGTTTTCGACCTTGAGACGTAGTGTGACGCGATCGGCATAACCACCACGCT5280 CATCGATAATTTCACCGCCGAAAGGCGCGGTGCCGCTGGCGACCTGCGTTTCACCCTGCC5340 ATAAAGAAACTGTTACCCGTAGGTAGTCACGCAACTCGCCGCACATCTGAACTTCAGCCT5400 CCAGTACAGCGCGGCTGAAATCATCATTAAAGCGAGTGGCAACATGGAAATCGCTGATTT5460 GTGTAGTCGGTTTATGCAGCAACGAGACGTCACGGAAAATGCCGCTCATCCGCCACATAT5520 CCTGATCTTCCAGATAACTGCCGTCACTCCAACGCAGCACCATCACCGCGAGGCGGTTTT5580 CTCCGGCGCGTAAAAATGCGCTCAGGTCAAATTCAGACGGCAAACGACTGTCCTGGCCGT5640 AACCGACCCAGCGCCCGTTGCACCACAGATGAAACGCCGAGTTAACGCCATCAAAAATAA5700 TTCGCGTCTGGCCTTCCTGTAGCCAGCTTTCATCAACATTAAATGTGAGCGAGTAACAAC5760 CCGTCGGATTCTCCGTGGGAACAAACGGCGGATTGACCGTAATGGGATAGGTTACGTTGG5820 TGTAGATGGGCGCATCGTAACCGTGCATCTGCCAGTTTGAGGGGACGACGACAGTATCGG5880 CCTCAGGAAGATCGCACTCCAGCCAGCTTTCCGGCACCGCTTCTGGTGCCGGAAACCAGG5940 CAAAGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCT6000 CTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAA6060 CGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGGATCCCTCGAGGAATTCATTT6120 ATAGCATAGAAAAAAACAAAATGAAATTCTACTATATTTTTACATACATATATTCTAAAT6180 ATGAAAGTGGTGATTGTGACTAGCGTAGCATCGCTTCTAGACATATACTATATAGTAATA6240 CCAATACTCAAGACTACGAAACTGATACAATCTCTTATCATGTGGGTAATGTTCTCGATG6300 TCGAATAGCCATATGCCGGTAGTTGCGATATACATAAACTGATCACTAATTCCAAACCCA6360 CCCGCTTTTTATAGTAAGTTTTTCACCCATAAATAATAAATACAATAATTAATTTCTCGT6420 AAAAGTAGAAAATATATTCTAATTTATTGCACGGTAAGGAAGTAGAATCATAAAGAACAG6480 TGACGGATCCCGGGATGGCCACACAGGGACAACGCGTCAACTGGGGAGATGAACCTTCCA6540 AAAGACGTGGTCGTTCTAACTCTCGTGGTCGGAAGAATAATGATATACCTTTGTCATTCT6600 ACAACCCCATTACCCTCGAACAAGGATCTAAATTTTGGAATTTATGTCCGAGAGACCTTG6660 TTCCCAAAGGAATAGGTAATAAGGATCAACAAATTGGTTATTGGAATAGACAGATTCGTT6720 ATCGTATTGTAAAAGGCCAGCGTAAGGAACTCGCTGAGAGGTGGTTCTTTTACTTCTTAG6780 GTACAGGACCTCATGCTGATGCTAAATTCAAAGACAAGATTGATGGAGTCTTCTGGGTTG6840 CAAGGGATGGTGCCATGAACAAGCCCACAACGCTTGGCACTCGTGGAACCAATAACGAAT6900 CCAAACCACTGAGATTTGATGGTAAGATACCGCCACAGTTTCAGCTTGAAGTGAACCGTT6960 CTAGGAACAATTCAAGGTCTGGTTCTCAGTCTAGATCTGTTTCAAGAAACAGATCTCAAT7020 CTAGAGGAAGACACCATTCCAATAACCAGAATAATAATGTTGAGGATACAATTGTAGCCG7080 TGCTTGAAAAATTAGGTGTTACTGACAAACAAAGGTCACGTTCTAAACCTAGAGAACGTA7140 GTGATTCCAAACCTAGGGACACAACACCTAAGAATGCCAACAAACACACCTGGAAGAAAA7200 CTGCAGGCAAGGGAGATGTGACAACTTTCTATGGTGCTAGAAGTAGTTCAGCTAACTTTG7260 GTGATAGTGATCTCGTTGCCAATGGTAACGCTGCCAAATGCTACCCTCAGATAGCTGAAT7320 GTGTTCCATCAGTGTCTAGCATAATCTTTGGCAGTCAATGGTCTGCTGAAGAAGCTGGTG7380 ATCAAGTGAAAGTCACGCTCACTCACACCTACTACCTGCCAAAGGATGATGCCAAAACTA7440 GTCAATTCCTAGAACAGATTGACGCTTACAAGCGACCTTCTGAAGTGGCTAAGGATCAGA7500 GGCAAAGAAGATCCCGTTCTAAGTCTGCTGATAAGAAGCCTGAGGAGTTGTCTGTAACTC7560 TTGTGGAGGCATACACAGATGTGTTTGATGACACACAGGTTGAGATGATTGATGAGGTTA7620 CGAACTAAACGCATGCCCGGGAATTCTGTGAGCGTATGGCAAACGAAGGAAAAATTAGTT7680 ATAGTAGCCGCACTCGATGGGACATTTCAACGTAAACCGTTTAATAATATTTTGAATCTT7740 ATTCCATTATCTGAAATGGTGGTAAAACTAACTGCTGTGTGTATGAAATGCTTTAAGGAG7800 GCTTCCTTTTCTAAACGATTGGGTGAGGAAACCGAGATAGAAATAATAGGAGGTAATGAT7860 ATGTATCAATCGGTGTGTAGAAAGTGTTACATCGACTCATAATATTATATTTTTTATCTA7920 AAAAACTAAAAATAAACATTGATTAAATTTTAATATAATACTTAAAAATGGATGTTGTGT7980 CGTTAGATAAACCGTTTATGTATTTTGAGGAAATTGATAATGAGTTAGATTACGAACCAG8040 AAAGTGCAAATGAGGTCGCAAAAAAACTGCCGTATCAAGGACAGTTAAAACTATTACTAG8100 GAGAATTATTTTTTCTTAGTAAGTTACAGCGACACGGTATATTAGATGGTGCCACCGTAG8160 TGTATATAGGATCTGCTCCCGGTACACATATACGTTATTTGAGAGATCATTTCTATAATT8220 TAGGAGTGATCATCAAATGGATGCTAATTGACGGCCGCCATCATGATCCTATTTTAAATG8280 GATTGCGTGATGTGACTCTAGTGACTCGGTTCGTTGATGAGGAATATCTACGATCCATCA8340 AAAAACAACTGCATCCTTCTAAGATTATTTTAATTTCTGATGTGAGATCCAAACGAGGAG8400 GAAATGAACCTAGTACGGCGGATTTACTAAGTAATTACGCTCTACAAAATGTCATGATTA8460 GTATTTTAAACCCCGTGGCGTCTAGTCTTAAATGGAGATGCCCGTTTCCAGATCAATGGA8520 TCAAGGACTTTTATATCCCACACGGTAATAAAATGTTACAACCTTTTGCTCCTTCATATT8580 CAGGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCG8640 CCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCG8700 CCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCTT8760 TACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACCATCTGCTCTGA8820 TGCCGCATAGTTAAGCCAGTACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCC8880 CCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGC8940 TTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATC9000 ACCGAAACGCGCGAGGCAG9019 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 262 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Feline infectious peritonitis virus (vii) IMMEDIATE SOURCE: (B) CLONE: FIPV E1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: MetLysTyrIleLeuLeuIleLeuAlaCysIleIleAlaCysValTyr 151015 GlyGluArgTyrCysAlaMetGlnAspSerGlyLeuGlnCysIleAsn 202530 GlyThrAsnSerArgCysGlnThrCysPheGluArgGlyAspLeuIle 354045 TrpHisLeuAlaAsnTrpAsnPheSerTrpSerValIleLeuIleVal 505560 PheIleThrValLeuGlnTyrGlyArgProGlnPheSerTrpLeuVal 65707580 TyrGlyIleLysMetLeuIleMetTrpLeuLeuTrpProIleValLeu 859095 AlaLeuThrIlePheAsnAlaTyrSerGluTyrGlnValSerArgTyr 100105110 ValMetPheGlyPheSerValAlaGlyAlaValValThrPheAlaLeu 115120125 TrpMetMetTyrPheValArgSerValGlnLeuTyrArgArgThrLys 130135140 SerTrpTrpSerPheAsnProGluThrAsnAlaIleLeuCysValAsn 145150155160 AlaLeuGlyArgSerTyrValLeuProLeuAspGlyThrProThrGly 165170175 ValThrLeuThrLeuLeuSerGlyAsnLeuTyrAlaGluGlyPheLys 180185190 MetAlaGlyGlyLeuThrIleGluHisLeuProLysTyrValMetIle 195200205 AlaThrProSerArgThrIleValTyrThrLeuValGlyLysGlnLeu 210215220 LysAlaThrThrAlaThrGlyTrpAlaTyrTyrValLysSerLysAla 225230235240 GlyAspTyrSerThrGluAlaArgThrAspAsnLeuSerGluHisGlu 245250255 LysLeuLeuHisMetVal 260 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 377 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Feline infectious peritonitis virus (vii) IMMEDIATE SOURCE: (B) CLONE: FIPV N (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: MetAlaThrGlnGlyGlnArgValAsnTrpGlyAspGluProSerLys 151015 ArgArgGlyArgSerAsnSerArgGlyArgLysAsnAsnAspIlePro 202530 LeuSerPheTyrAsnProIleThrLeuGluGlnGlySerLysPheTrp 354045 AsnLeuCysProArgAspLeuValProLysGlyIleGlyAsnLysAsp 505560 GlnGlnIleGlyTyrTrpAsnArgGlnIleArgTyrArgIleValLys 65707580 GlyGlnArgLysGluLeuAlaGluArgTrpPhePheTyrPheLeuGly 859095 ThrGlyProHisAlaAspAlaLysPheLysAspLysIleAspGlyVal 100105110 PheTrpValAlaArgAspGlyAlaMetAsnLysProThrThrLeuGly 115120125 ThrArgGlyThrAsnAsnGluSerLysProLeuArgPheAspGlyLys 130135140 IleProProGlnPheGlnLeuGluValAsnArgSerArgAsnAsnSer 145150155160 ArgSerGlySerGlnSerArgSerValSerArgAsnArgSerGlnSer 165170175 ArgGlyArgHisHisSerAsnAsnGlnAsnAsnAsnValGluAspThr 180185190 IleValAlaValLeuGluLysLeuGlyValThrAspLysGlnArgSer 195200205 ArgSerLysProArgGluArgSerAspSerLysProArgAspThrThr 210215220 ProLysAsnAlaAsnLysHisThrTrpLysLysThrAlaGlyLysGly 225230235240 AspValThrThrPheTyrGlyAlaArgSerSerSerAlaAsnPheGly 245250255 AspSerAspLeuValAlaAsnGlyAsnAlaAlaLysCysTyrProGln 260265270 IleAlaGluCysValProSerValSerSerIleIlePheGlySerGln 275280285 TrpSerAlaGluGluAlaGlyAspGlnValLysValThrLeuThrHis 290295300 ThrTyrTyrLeuProLysAspAspAlaLysThrSerGlnPheLeuGlu 305310315320 GlnIleAspAlaTyrLysArgProSerGluValAlaLysAspGlnArg 325330335 GlnArgArgSerArgSerLysSerAlaAspLysLysProGluGluLeu 340345350 SerValThrLeuValGluAlaTyrThrAspValPheAspAspThrGln 355360365 ValGluMetIleAspGluValThrAsn 370375 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid (vi) ORIGINAL SOURCE:

(A) ORGANISM: Feline infectious peritonitis virus (vii) IMMEDIATE SOURCE: (B) CLONE: N primer #1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CTCGTGGTCGGAAGAATAATGATA24 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid (vi) ORIGINAL SOURCE: (A) ORGANISM: Feline infectious peritonitis virus (vii) IMMEDIATE SOURCE: (B) CLONE: N primer #2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: AGCACCATAGAAAGTTGTCACATC24 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid (vi) ORIGINAL SOURCE: (A) ORGANISM: Feline infectious peritonitis virus (vii) IMMEDIATE SOURCE: (B) CLONE: E1 primer #1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: TATGTAATGTTCGGCTTTAGTG22 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid (vi) ORIGINAL SOURCE: (A) ORGANISM: Feline infectious peritonitis virus (vii) IMMEDIATE SOURCE: (B) CLONE: E1 primer #2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: GTGCTTCTGTTGAGTAATCACC22 __________________________________________________________________________

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